Professional Documents
Culture Documents
Pohanka, Adam, Kizek, 2013
Pohanka, Adam, Kizek, 2013
3390/s130911498
OPEN ACCESS
sensors
ISSN 1424-8220
www.mdpi.com/journal/sensors
Article
2
3
Sensors 2013, 13
11499
1. Introduction
Nerve agents are a group of neurotoxic compounds having common molecular mechanism of
action. They are irreversible inhibitors of the enzyme acetylcholinesterase (AChE; EC 3.1.1.7). It is
noteworthy that the enzyme plays a crucial role in cholinergic neurotransmission [1,2]. The nerve
agents are probably the most toxic chemical substances used for chemical warfare. Their median lethal
doses extrapolated for humans are much lower than the doses for blister or blood agents [1,3].
Organophosphate and carbamate pesticides have similar mechanisms of action, because they can also
inhibit AChE by irreversible (organophosphates) and pseudo-irreversible (carbamates) mechanisms of
action [2,4].
The fact that AChE can be inhibited by neurotoxic compounds is not only a negative phenomenon.
Firstly, many drugs act as inhibitors of AChE. Secondly, the phenomenon can be used in analytical
chemistry. The activity of AChE can easily be measured using disparate colorimetric, fluorimetric and
voltammetric protocols [5]. When the measurement is performed in vitro, it can be used for an assay of
the aforementioned inhibitors [6]. The assay can be done using standard cuvettes, microplates, etc.
Besides the aforementioned, it is well suited for the construction of biosensors that are miniaturized
devices containing a physico-chemical transducer in a tight junction with a biorecognition element, in
this case represented by the AChE [7,8].
The intention in the present experiments was to develop a biosensor to assay highly toxic nerve
agents. For this purpose, a novel immobilization procedure based on capturing of AChE into a gelatin
membrane and consequent stabilization by glutaraldehyde was chosen. Nerve agents are not easy to
detect prior to the appearance of human casualties when misused in chemical warfare or
chemical terrorism. Experiences from the Tokyo subway attack point to the necessity of fast
countermeasures [9,10]. Detection of the agents is needed for initiation of the provision of suitable
prophylactic and therapeutic care. We have thus aimed our efforts at constructing a cheap and reliable
device suitable for both laboratory and field assays.
2. Experimental Section
2.1. Preparation of the Biosensor
Lyophilized electric eel AChE (specific activity 16.7 kat/mg protein) was purchased from
Sigma-Aldrich (Saint Louis, MO, USA). The enzyme was dissolved in phosphate buffered saline prior
to assay. In order to specify and adjust the amount of enzyme, AChE activity was measured by a
modified Ellmans method in a way introduced in previous papers [1113].
Screen printed sensors were received from BVT Technologies (Brno, Czech Republic). The sensors
are depicted in Figure 1. We chose sensors sized 25.4 7.3 0.6 mm containing a central platinum dot
shaped working electrode with diameter 1 mm, platinum auxiliary and a reference electrode consisting
of silver covered with silver chloride. We chose the platinum working electrodes in compliance with
our good previous experience working with this material and data found in the literature [1417]. The
working electrode was covered with 10 L of a solution containing AChE 20 U for 1 mmol/L
acetylthiocholine as a substrate, gelatin 1 mg/mL (Sigma-Aldrich) and 1% (w/v) glutardialdehyde
(Sigma-Aldrich). The solution was dried at laboratory temperature overnight. After that, surface of the
Sensors 2013, 13
11500
biosensor was rinsed with phosphate buffered saline and dried at laboratory temperature. The freshly
prepared biosensors were used immediately or kept stored at 4 C until needed.
Figure 1. Screen printed sensors used in the experiments. In the upper part, sensor in a
plastic holder is depicted. In the bottom part, a naked sensor is shown. Electrodes are on
the left side of the picture, output contacts on the right.
Sensors 2013, 13
11501
Figure 2. Structures of nerve agents used for the experiment.
Sensors 2013, 13
11502
necessary for an easy assay. Compared to this, the distribution and elimination processes in the human
body are not identical to the environment in the reaction cell and membrane on the biosensor surface.
Figure 3. Calibration of AChE based biosensors to the tested nerve agents sarin (A),
soman (B), tabun (C) and VX (D). Error bars indicate standard deviation for n = 4 and green
lines indicate the 95% confidence interval. The point in brackets was achieved by a
blank assay.
A
600
500
600
2
( )
500
R =0.997
300
300
200
200
100
100
0
-12
-10
-8
-6
-4
-12
log[sarin] (mol/L)
-10
-8
-6
-4
log[soman] (mol/L)
D
600
500
600
2
( )
500
R =0.991
400
( )
R =0.993
400
i (nA)
i (nA)
R =0.996
400
i (nA)
i (nA)
400
( )
300
300
200
200
100
100
-12
-10
-8
log[tabun] (mol/L)
-6
-4
-12
-10
-8
-6
-4
log[VX] (mol/L)
The results indicate a substantial advantage of AChE-based biosensors to assay nerve agents
without the need for any expensive device or complicated protocol. Though the biosensors have some
disadvantages, especially in their unsuitability for automated long term monitoring, the assays can be
easily performed under field conditions and they are quite cheap compared to physical detectors such
as mass spectrometers [21,22]. Nerve agents can be also detected by colorimetric devices with scoring
by a naked eye [23]. Paraoxonase-based colorimetric biosensors are another option [24,25]. Overall the
simplicity and easy interpretation of data is a major advantage of the colorimetric devices. On the other
hand, the electrochemical protocol proposed here allows one to reach lower limits of detection.
Electrochemical detection of nerve agents can be further used for miniaturization and partially
automated assay procedures [26]. The limits of detection reached by our biosensor are close to the
limit of detection of organophosphate and carbamate pesticide detections described in papers devoted
Sensors 2013, 13
11503
to AChE-based electrochemical biosensors [27], bulk acoustic biosensors [25] and surface plasmon
resonance biosensors [28]. Arduini and co-workers used a butyrylcholinesterase based electrochemical
biosensor and analysed sarin and VX with detection limits of 12 and 14 ppb [29]. Schulze and
co-workers reached even lower limits of detection than we did when they used a protein engineered
AChE [30]. In an example, Schulze and co-workers detected pirimiphos in a concentration of
3.5 1012 mol/L. Their results are also interesting due to fact that they analysed pesticides with lower
affinity (lower bimolecular rate constant) toward the enzyme. Comparing with their work, we analysed
highly reactive compounds. Limitations for successful assay of nerve agents result from the lack of
ability to diffuse through a membrane rather than the affinity toward AChE, which is high enough.
It should be emphasized that the biosensor was used for assay of highly toxic nerve agents that are
not readily available to most researchers. In the current scientific literature, AChE-based biosensors are
dominantly used for the more widely available pesticides. The results presented here are unique for
that reason. Moreover, an immobilization procedure using gelatin in combination with gluataraldehyde
stabilization was used. The biosensor construction procedure was successful and worthy of use as a
platform for standard production of biosensors.
The AChE-based biosensor is not a selective analytical tool. In the assay, all neurotoxic compounds
acting as AChE inhibitors, including some Alzheimer disease drugs, carbamates and oxoforms of
organophosphate pesticides can be assayed and the compounds will be active in the assay once
presented in a sample. On the other hand, these compounds cause similar toxicological manifestations
as the nerve agents and they are also harmful for humans when present in high levels [2,9,3133].
Organic solvents can be considered as more serious interference problem as they can be anticipated in
a sample [34,35] and they are not toxic enough to be considered as a menace. Ethanol and methanol
are typical examples of such organic solvents [36]. Immobilization of AChE causes its stabilization
and better resistance to organic solvents than typical for the free enzyme [37]. It is a problem because
the solvents can be easily considered as suspicious samples or be used for processing samples such as
in the extraction of aflatoxins that inhibit AChE as well [38,39]. We tested the interference of ethanol,
methanol, isopropanol, and acetonitrile using the prepared biosensors. This was tested when the
organic solvent when either applied as a pure sample (100 L) resulting in a final concentration in the
cuvette of 10% (v/v) and then with a double volume of sample (200 L) by lowering the volume of g
phosphate buffered saline from the original 800 to 700 L. Percent of inhibition was calculated using a
control measurement when phosphate buffered saline was used as a sample. The results are listed in
Table 1. We can see that isopropanol caused the lowest inhibition and it appear to be the best for
sample processing. Ethanol, methanol and acetonitrile can cause significant inhibition. However, the
assay of nerve agents is still functional as there remains a lot of AChE activity on the biosensor.
Pertinent increase of sample volume could lead to interference of organic solvent so a maximum
sample volume of 100 L is recommended for the 1 mL cuvette. The findings correlate well to results
from known papers [5,13,37].
Sensors 2013, 13
11504
Ethanol
Methanol
Isopropanol
Acetonitrile
31.6 3.8
49.8 4.1
37.2 2.7
45.6 3.9
17.4 2.1
34.9 5.6
29.8 3.5
51.4 5.2
4. Conclusions
We can conclude that the prepared biosensor is suitable for a routine, low cost field assay of nerve
agents. There is no need to perform any elaborate manipulation of the samples. The biosensor is
reliable, with quite low sensitivity to interference. In the assay, the overall costs are low and the
biosensor seems to be amenable to large-scale production.
Acknowledgments
Financial support from CEITEC CZ.1.05/1.1.00/02.0068 is highly acknowledged. A long-term
organization development plan 1011 (Faculty of Military Health Sciences, University of Defence,
Czech Republic) is acknowledged as well.
Conflicts of Interest
The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Sensors 2013, 13
11505
10. Furtado, M.D.; Rossetti, F.; Chanda, S.; Yourick, D. Exposure to nerve agents: From status
epilepticus to neuroinflammation, brain damage, neurogenesis and epilepsy. Neurotoxicology
2012, 33, 14761490.
11. Ellman, G.L.; Courtney, K.D.; Andres, V., Jr.; Feather-Stone, R.M. A new and rapid colorimetric
determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 8895.
12. Pohanka, M. Acetylcholinesterase based dipsticks with indoxylacetate as a substrate for assay of
organophosphates and carbamates. Anal. Lett. 2012, 45, 367374.
13. Pohanka, M.; Fusek, J.; Adam, V.; Kizek, R. Carbofuran assay using gelatin based biosensor with
acetylcholinesterase as a recognition element. Int. J. Electrochem. Sci. 2013, 8, 7179.
14. Turdean, G.L.; Popescu, I.C.; Oniciu, L.; Thevenot, D.R. Sensitive detection of organophosphorus
pesticides using a needle type amperometric acetylcholinesterase-based bioelectrode. Thiocholine
electrochemistry and immobilised enzyme inhibition. J. Enzyme Inhib. Med. Chem. 2002, 17,
107115.
15. Yang, L.; Wang, G.; Liu, Y. An acetylcholinesterase biosensor based on platinum
nanoparticles-carboxylic graphene-nafion-modified electrode for detection of pesticides.
Anal. Biochem. 2013, 437, 144149.
16. Jeanty, G.; Wojciechowska, A.; Marty, J.L.; Trojanowicz, M. Flow-injection amperometric
determination of pesticides on the basis of their inhibition of immobilized acetylcholinesterases of
different origin. Anal. Bioanal. Chem. 2002, 373, 691695.
17. Bucur, M.P.; Bucur, B.; Radu, G.L. Critical evaluation of acetylcholine iodide and
acetylthiocholine chloride as substrates for amperometric biosensors based on acetylcholinesterase.
Sensors 2013, 13, 16031613.
18. Hubaux, A.; Vos, G. Decision and detection limits for linear calibration curves. Anal. Chem. 1970,
42, 849855.
19. Luo, W.; Li, H.; Zhang, Y.; Ang, C.Y.W. Determination of formaldehyde in blood plasma by
high-performance liquid chromatography with fluorescence detection. J. Chromatogr. B 2001,
753, 253257.
20. Liu, S.Q.; Zheng, Z.Z.; Li, X.Y. Advances in pesticide biosensors: current status, challenges, and
future perspectives. Anal. Bioanal. Chem. 2013, 405, 6390.
21. Seto, Y.; Kanamori-Kataoka, M.; Tsuge, K.; Ohsawa, I.; Iura, K.; Itoi, T.; Sekiguchi, H.;
Matsushita, K.; Yamashiro, S.; Sano, Y.; et al. Sensitive monitoring of volatile chemical warfare
agents in air by atmospheric pressure chemical ionization mass spectrometry with counter-flow
introduction. Anal. Chem. 2013, 85, 26592666.
22. Mwila, K.; Burton, M.H.; Van Dyk, J.S.; Pletschke, B.I. The effect of mixtures of
organophosphate and carbamate pesticides on acetylcholinesterase and application of
chemometrics to identify pesticides in mixtures. Environ. Monit. Assess. 2013, 185, 23152327.
23. Vymazalova, K.; Halamek, E.; Kadlcak, J. Photocolorimetric biosensor for detection of
cholinergic organophosphorus compounds. Def. Sci. J. 2012, 62, 399403.
24. Chen, C.H.; Yang, K.L. A liquid crystal biosensor for detecting organophosphates through the
localized pH changes induced by their hydrolytic products. Sens. Actuators B-Chem. 2013, 181,
368374.
Sensors 2013, 13
11506
25. Chen, D.; Wang, J.J.; Xu, Y.; Li, D.H.; Zhang, L.Y.; Li, Z.X. Highly sensitive detection of
organophosphorus pesticides by acetylcholinesterase-coated thin film bulk acoustic resonator
mass-loading sensor. Biosens. Bioelectron. 2013, 41, 163167.
26. Upadhyay, L.S.B.; Verma, N. Enzyme inhibition based biosensors: A review. Anal. Lett. 2013, 46,
225241.
27. Arduini, F.; Guidone, S.; Amine, A.; Palleschi, G.; Moscone, D. Acetylcholinesterase biosensor
based on self-assembled monolayer-modified gold-screen printed electrodes for
organophosphorus insecticide detection. Sens. Actuators B-Chem. 2013, 179, 201208.
28. Huang, X.; Tu, H.Y.; Zhu, D.H.; Du, D.; Zhang, A.D. A gold nanoparticle labeling strategy for
the sensitive kinetic assay of the carbamate-acetylcholinesterase interaction by surface plasmon
resonance. Talanta 2009, 78, 10361042.
29. Arduini, F.; Amine, A.; Moscone, D.; Ricci, F.; Palleschi, G. Fast, sensitive and cost-effective
detection of nerve agents in the gas phase using a portable instrument and an electrochemical
biosensor. Anal. Bioanal. Chem. 2007, 388, 10491057.
30. Schulze, H.; Muench, S.B.; Villatte, F.; Schmid, R.D.; BAchmann, T.T. Insecticide detection
through protein engineering of nippostrongylus brasiliensis acetylcholinesterase B. Anal. Chem.
2005, 77, 58235830.
31. Bartolini, M.; Cavrini, V.; Andrisano, V. Characterization of reversible and pseudo-irreversible
acetylcholinesterase inhibitors by means of an immobilized enzyme reactor. J. Chromatogr. A
2007, 1144, 102110.
32. de los Rios, C. Cholinesterase inhibitors: A patent review (20072011). Expert Opin. Ther. Pat.
2012, 22, 853869.
33. Holzgrabe, U.; Kapkova, P.; Alptuzun, V.; Scheiber, J.; Kugelmann, E. Targeting
acetylcholinesterase to treat neurodegeneration. Expert. Opin. Ther. Tar. 2007, 11, 161179.
34. Pietsch, M.; Christian, L.; Inhester, T.; Petzold, S.; Gutschow, M. Kinetics of inhibition of
acetylcholinesterase in the presence of acetonitrile. FEBS J. 2009, 276, 22922307.
35. Turdean, G.L.; Turdean, M.S. Synergetic effect of organic solvents and paraoxon on the
immobilized acetylcholinesterase. Pest. Biochem. Physiol. 2008, 90, 7381.
36. Fekonja, O.; Zorec-Karlovsek, M.; El Kharbili, M.; Fournier, D.; Stojan, J. Inhibition and
protection of cholinesterases by methanol and ethanol. J. Enzyme Inhib. Med. Chem. 2007, 22,
407415.
37. Mionetto, N.; Marty, J.L. Acetylcholinestrase in organic-solvents for detection of
pesticides - biosensors application. Biosens. Bioelectron. 1994, 9, 463470.
38. Sheijooni-Fumani, N.; Hassan, J.; Yousefi, S.R. Determination of aflatoxin B1 in cereals by
homogeneous
liquid-liquid
extraction
coupled
to
high
performance
liquid
chromatography-fluorescence detection. J. Sep. Sci. 2011, 34, 13331337.
39. Pohanka, M. Spectrophotomeric assay of aflatoxin B1 using acetylcholinesterase immobilized on
standard microplates. Anal. Lett. 2013, 46, 13061315.
2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).